1. Field of the Invention
The present invention relates generally to the fields of molecular biology and nucleic acid sequencing. More particularly, it concerns methods of barcoding nucleic acids.
2. Description of Related Art
Barcodes can be used to identify nucleic acid molecules, for example, where sequencing can reveal a certain barcode coupled to a nucleic acid molecule of interest. In some instances, a sequence-specific event can be used to identify a nucleic acid molecule, where at least a portion of the barcode is recognized in the sequence-specific event, e.g., at least a portion of the barcode can participate in a ligation or extension reaction. The barcode can therefore allow identification, selection or amplification of gDNA molecules that are coupled thereto.
One method to couple a barcode to nucleic acid molecules of interest includes preparation of an Ion gDNA fragment library, as described by Life Technologies for the Ion Torrent System. In this method, fragments of gDNA are ligated to adaptors, where at least one end of each fragment of genomic DNA is ligated to an adaptor including a barcode. The ligated adaptors and gDNA fragments may be nick repaired, size selected, and amplified using PCR with primers directed to the adaptors to produce an amplified library. For example, ligation adaptors including 16 different barcodes can be used to prepare 16 different gDNA samples, each with a unique barcode, such that either each sample can be amplified separately by PCR using the same PCR primers and then pooled (mixed together) or each sample can be pooled first and then simultaneously amplified using the same PCR primers. As a result each gDNA sample can be identified by its attached unique barcode. However, the number of different ligation adaptors needed is equal to the number of barcodes. For example, the production of 256 sample libraries capable of being sequenced as a mixture would require 256 different ligation adapters.